As shown in FIG. 1, diaminopimelate (DAP) decarboxylase, (EC 4.1.1.20), acts in the last step in the lysine biosynthetic pathway, by catalyzing the decarboxylation of meso-diaminopimelate to lysine. In bacteria, DAP decarboxylase plays a crucial role in balancing two essential metabolites; meso-DAP, a component of cell wall peptidoglycan (and a spore wall component), and the amino acid lysine, used for protein production. DAP decarboxylases isolated from both plants and bacteria have a strict requirement for pyridoxal-phosphate (PLP) as a cofactor. DAP decarboxylase is the only PLP-dependent alpha-decarboxylase known to work on a D-amino acid.
Commercially, L-lysine is primarily used as a supplement for animal feeds derived from grains that contain only limited quantities of this amino acid. Poultry, swine, and other livestock are unable to synthesize L-lysine and therefore must have this amino acid supplied as part of their diet. Currently, L-lysine is manufactured by either direct fermentation or enzymatic conversion of DL-.alpha.-amino-.epsilon.-caprolactam. Fermentation processes using strains of Corynebacterium glutamicum or Brevibacterium lactofermentum have become the major methods for L-lysine production. (O. Tosaka et al., Trends in Biotechnology, 1, 70 (1986)).
Recently, F. J. Schendel et al. in J. Appl. Environ. Microbiol., 56, 963 (1990), identified homoserine auxotrophs and S-(2-aminoethyl)-cysteine (AEC) resistant mutants of a thermophilic methylotrophic Bacillus sp. which overproduce significant quantities of L-lysine at 50.degree. C. Such thermophilic methylotrophs may have advantages over other organisms for industrial use, as discussed by Al-Awadhi et al., Biotechnol. Bioeng., 36, 816, 821 (1990). In particular, the methylotrophic Bacillus MGA3 identified by F. J. Schendel et al., cited supra, may have significant advantages over other bacilli for the overproduction of lysine since it does not sporulate at high temperatures even under conditions of nutrient limitation, in contrast to lysine-producing mutants of B. licheniformis that sporulated when grown at temperatures greater than 40.degree. C. (H. Hagino et al., Biotechnol. Lett., 3, 425 (1981)).
Since both spore components, diaminopimelate and dipicolinic acid, are derived from the lysine biosynthetic pathway, as shown in FIG. 1, differences in the regulation of this pathway may occur between this thermophilic Bacillus sp. and other mesophilic bacilli. Therefore, a need exists to isolate and characterize the informational macromolecules (DNA and RNA) which function in the biosynthetic pathway to lysine, methionine and threonine in the thermotolerant Bacillus sp. MGA3. A further need exists to isolate and characterize the products, such as the enzymes, that function in these biosynthetic pathways. A further need exists to produce mutant varieties of said informational macromolecules, in order to improve the properties of the enzymes and other polypeptides encoded thereby, or to produce improved strains of thermophilic, methylotrophic bacteria.